Selective estrogen receptor modulators differentially alter the immune response of gilthead seabream juveniles.

17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives and hormone replacement therapy, tamoxifen (Tmx), a selective estrogen-receptor modulator used in hormone replacement therapy, and G1, a G protein-coupled estrogen receptor (GPER) selective agonist, differentially increased the hepatic vitellogenin (vtg) gene expression and altered the immune response in adult gilthead seabream (Sparus aurata L.) males. However, no information exists on the effects of these compounds on the immune response of juveniles. This study aims, for the first time, to investigate the effects of the dietary intake of EE2, Tmx or G1 on the immune response of gilthead seabream juveniles and the capacity of the immune system of the specimens to recover its functionality after ceasing exposures (recovery period). The specimens were immunized with hemocyanin in the presence of aluminium adjuvant 1 (group A) or 120 (group B) days after the treatments ceased (dpt). The results indicate that EE2 and Tmx, but not G1, differentially promoted a transient alteration in hepatic vtg gene expression. Although all three compounds did not affect the production of reactive oxygen intermediates, they inhibited the induction of interleukin-1ß (il1b) gene expression after priming. Interestingly, although Tmx increased the percentage of IgM-positive cells in both head kidney and spleen during the recovery period, the antibody response of vaccinated fish varied depending on the compound used and when the immunization was administered. Taken together, our results suggest that these compounds differentially alter the capacity of fish to respond to infection during ontogeny and, more interestingly, that the adaptive immune response remained altered to an extent that depends on the compound.

Richness and diversity of helminth species in eels from a hypersaline coastal lagoon, Mar Menor, south-east Spain.

The composition and diversity of parasite communities and intestinal components, as well as infra-community structure, were assessed in eels Anguilla anguilla, from Mar Menor, a permanent Mediterranean hypersaline coastal lagoon. Data were used to determine whether this helminth community differs in composition and structure from that of eels in lagoons with lower salinity regimes and higher freshwater inputs. A total prevalence of 93% was detected. Specifically, parasites were identified as Deropristis inflata, Bucephalus anguillae, Contracaecum sp., Anguillicoloides crassus and two plerocercoid larvae belonging to the order Proteocephalidae, the marine species representing 91% of the isolated helminths. In the total community, digenetic trematodes were the dominant group of helminths, and D. inflata, an eel specialist, dominated both the component community and the infra-community. Richness and diversity were low but similar to those reported in other saline lagoons, and maximum species per eel did not exceed four. At the infra-community level, higher abundance than in other brackish or marine Mediterranean environments was detected. The findings provide further evidence of the similarity in composition and structure of helminth communities in eels from various Mediterranean coastal lagoons. Moreover, salinity-dependent specificities are well supported and reflect the life history of individual eels.

A role for matrix metalloproteinases in granulocyte infiltration and testicular remodelation in a seasonal breeding teleost.

The testicular cystic structure and the abrupt morphological changes that the fish testis undergoes during the reproductive cycle (RC) make it an interesting model for studying the regulation of spermatogenesis, in particular the role of matrix metalloproteinases (Mmps). The gilthead seabream is a seasonal breeding teleost whose testis undergoes drastic remodeling events, especially during the post-spawning stage when a massive infiltration of a immune cell type, the acidophilic granulocytes, occurred. Bearing this in mind, we studied the gilthead seabream testis gelatinolytic activities involved in migration and tissue remodeling and its regulation by endocrine, immune and tissue stimuli. Thus, we demonstrated that the germinal epithelium of the testis showed gelatinolytic activity during spermatogenesis and post-spawning but not during resting, when only scarce interstitial cells were stained. Moreover, the precursor and mature forms of two gelatinases, Mmp2- and Mmp9-like, were active in the gonad, whose activities were up-regulated by 17beta-estradiol (E(2)) but not by lipopolysaccharide (LPS)/bacterial DNA (VaDNA) in testicular cell suspensions. E(2) and LPS/VaDNA also up-regulated a variety of cytokines and chemokines. We also cloned mmp9, mmp13, tissue inhibitors of Mmps (timp)-2a and timp2b genes and found that all of them were expressed in the gonad in a RC stage-dependent manner. Interestingly, mmps and timps were highly expressed by the testicular acidophilic granulocytes. Moreover, in these cells, the gelatinolytic activity seemed to correspond to the precursor and mature forms of putative Mmp2 and Mmp9 gelatinases, while the main gelatinolytic activity seemed to correspond to the mature form of Mmp2 in head-kidney acidophilic granulocytes. Finally, although none of the stimuli used were able to induce the gelatinolytic activity of Mmp9-like in head-kidney acidophilic granulocytes, the expression of mmp9, timp2a and timp2b were all up-regulated by LPS/VaDNA in these cells, while only mmp9 and timp2a expression increased upon stimulation with gelatin.

Pattern of expression of immune-relevant genes in the gonad of a teleost, the gilthead seabream (Sparus aurata L.).

Immune responses in the testis are regulated in a way that provides protection for the developing male germ cells, while permitting qualitatively normal inflammatory responses and protection against infection. In addition, germ cells are potent targets for the growth factors and cytokines which regulate the reproductive process. Our study analyzes for the first time the pattern of expression of several immune-relevant genes in the gonad of a seasonal breeding teleost fish. The immune molecules analyzed include (i) inflammatory molecules, such as interleukin-1b (il1b), il6, tumor necrosis factor-a (tnfa), cyclooxygenase-2 (cox2) and the NADPH oxidase subunit p40(phox) (ncf4 gene); (ii) the anti-inflammatory cytokine transforming growth factor-b1 (tgfb1) and its type 2 receptor tgfbr2; (iii) innate immune receptors, including toll-like receptor 9 (tlr9), tlr5, tlr22 and macrophage-colony stimulating factor receptor (mcsfr); (iv) lymphocyte receptors, such as the beta subunit of T-cell receptor (Tcrb) and the heavy chain of immunoglobulin M (ighm); (v) the anti-bacterial molecules lysozyme (lyz), hepcidin (hamp) and complement component 3 (c3); (vi) the anti-viral molecule myxovirus (influenza) resistance protein (mx); and (vii) molecules related to leukocyte infiltration, including the CC chemokine ccl4, the CXC chemokine il8 and the leukocyte adhesion molecule E-selectin (Sele). Notably, all of them show a pattern of expression that depends on the reproductive stage of the first two reproductive cycles when the fish develop and function as males. Furthermore, we demonstrate that some of these immune-relevant molecules, such as Il1b and Mcsfr, are produced by germ cells (Il1b) and ovarian and testicular somatic cells (Mcsfr). These data suggest that, as occurs in mammals, there is a critical balance between immune molecules and that these may play an essential role in the orchestration of gametogenesis and the maintenance of gonad tissue homeostasis in fish.

Role of 5α-dihydrotestosterone in testicular development of gilthead seabream following finasteride administration.

In teleosts, spermatogenesis is regulated by pituitary gonadotropins and sex steroids. 5α-dihydrotestosterone (DHT), derived from testosterone (T) through the action of 5α-reductase, has recently been suggested to play a physiologically important role in some fish species. In this study, gilthead seabream, Sparus aurata L., males received an implant of 1µgT/g body mass (bm) or vehicle alone and, 7days later, 1mg finasteride (FIN, an inhibitor of 5α-reductase)/kg bm or vehicle. Serum levels of T, 11-ketotestosterone (11KT), DHT and 17ß-estradiol (E2), and the mRNA levels of the main enzymes involved in their synthesis, were analysed. T promoted a transient increase in the serum levels of T, 11KT and E2 but a decrease in those of DHT at day 15 following T injection, in accordance with the up-regulation of mRNA levels of the enzymes involved in T transformation to 11KT (coding genes: cyp11b1 and hsd11b) and the down-regulation of mRNA levels of the enzyme responsible for T transformation to DHT (coding gene: srd5a). Interestingly, a similar effect was observed when FIN was injected. However, when fish were injected with T and FIN successively (T+FIN), control levels were not recovered at the end of the experimental period (28days). DHT seems to regulate E2 serum levels via the down-regulation of mRNA levels of aromatase (coding gene: cyp19a1a), which is needed for the transformation of T into E2. The testis histology, together with the proliferative rates recorded upon T, FIN or T+FIN treatment, suggests that DHT is involved in the onset of the meiotic phase of spermatogenesis.

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination.

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.

17α-Ethynylestradiol alters the peritoneal immune response of gilthead seabream.

17α-Ethynylestradiol (EE2), a synthetic estrogen used in most oral contraceptives pills and hormone replacement therapies, is found in many water bodies, where it can modulate the fish immune response. EE2 acts as an endocrine disruptor in gilthead seabream, Sparus aurata L., a marine teleost fish of great economic value in Mediterranean aquaculture, as it induces hepatic vitellogenin gene (vtg) expression. Moreover, EE2 also alters the capacity of gilthead seabream to appropriately respond to infection although it does not behave as an immunosuppressor. Nevertheless, these previous studies have mainly focused on the head kidney leukocytes and no information exists on peritoneal leukocytes, including mast cells. In the present work, juvenile gilthead seabream fish were fed a pellet diet supplemented with EE2 for 76 days and intraperitoneally injected with hemocyanin plus imject alum adjuvant at the end of EE2 treatment and 92 days later, and the peritoneal immune response was analyzed. EE2 supplementation induced vtg expression but returned to basal levels by 3 months post-treatment. Interestingly, gilthead seabream peritoneal leukocytes express the genes encoding for the nuclear estrogen receptor α and the G protein-coupled estrogen receptor 1 and the dietary intake of EE2 induced these expression. Moreover, EE2 induced an inflammatory response in the peritoneal cavity in unvaccinated fish, which was largely maintained for several months after the cessation of the treatment. However, the impact of EE2 in vaccinated fish was rather minor and transient. Taken together, the study provides fresh information about endocrine immune disruption, focusing on peritoneal leukocytes.

Tamoxifen disrupts the reproductive process in gilthead seabream males and modulates the effects promoted by 17α-ethynylestradiol.

17α-Ethynylestradiol (EE2), which is used in oral contraceptives and hormone replacement therapy, is a well documented estrogenic endocrine disruptor and an aquatic contaminant. In the present study, adult male specimens of the marine hermaphrodite teleost gilthead (Sparus aurata L.) were fed a diet containing tamoxifen (Tmx), an estrogen receptor ligand used in cancer therapy, alone or combined with EE2, for 25 days and then fed a commercial diet for a further 25 days (recovery period). The effects of short (5days) and long (25 days) treatments on several reproductive and gonad immune parameters and the reversibility of the disruptive effects after the recovery period were examined. Our data showed that Tmx acted as an estrogenic endocrine disruptor as revealed by the increase in the hepatic transcription of the vitellogenin gene in males, the serum levels of 17ß-estradiol and the gonad expression levels of the estrogen receptor α and G protein-coupled estrogen receptor genes, and the recruitment of leukocytes into the gonad, a well known estrogenic-dependent process in gilthead seabream males. On the other hand, Tmx also increased sperm concentration and motility as well as the serum levels of androgens and the expression levels of genes that codify for androgenic enzymes, while decreasing the expression levels of the gene that code for gonadal aromatase. When applied simultaneously, Tmx and EE2 could act in synergy or counteract, each other, depending on the parameter measured. The disruptive effect of EE2 and/or Tmx was not reversible after a 25 day recovery period.

Tamoxifen persistently disrupts the humoral adaptive immune response of gilthead seabream (Sparus aurata L.).

There is increasing concern about the possible effect of pharmaceutical compounds may have on the fish immune system. Bath exposition of 17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives, altered the immune response of the gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. Tamoxifen (Tmx) is a selective estrogen-receptor modulator used in hormone replacement therapy, the effects of which are unknown in fish immunity. This study aims to investigate the effects of dietary administration of EE2 (5 µg/g food) and Tmx (100 µg/g food) on the immune response of gilthead seabream, and the capacity of the immune system to recover its functionality after a recovery period. The results show for the first time the reversibility of the effect of EE2 and Tmx on the fish immune response. Tmx promoted a transient alteration in hepatic vitellogenin gene expression of a different magnitude to that produced by EE2. Both, EE2 and Tmx inhibited the induction of interleukin-1ß gene expression while reversed the inhibition of ROI production in leukocytes following vaccination. However, none of these effects were observed after ceasing EE2 and Tmx exposure. EE2 and Tmx stimulated the antibody response of vaccinated fish although Tmx, but not EE2, altered the antibody response and modulated the percentage of IgM(+) B lymphocytes of vaccinated fish during the recovery phase. Taken together, our results suggest that EE2 and Tmx might alter the capacity of fish to appropriately respond to infection and show that Tmx has a long-lasting effect on humoral adaptive immunity.

A role for acidophilic granulocytes in the testis of the gilthead seabream (Sparus aurata L., Teleostei).

In mammals, a complex interaction between the immune and the reproductive systems has been described, in which testicular immune cells produce cytokines and growth factors which modulate gonad functions, while specific gonad cells influence the immune response in this organ. In this study we describe the presence of acidophilic granulocytes in the testis of the hermaphrodite teleost fish gilthead seabream (Sparus aurata L.) by using a specific monoclonal antibody. During the post-spawning stage of the testis, this cell type appears in the germinal compartment, accumulates interleukin (IL)-1beta and does not seem to be involved in the phagocytosis of degenerating cells. Moreover, in vitro, 11-ketotestosterone and 17beta-oestradiol, the principal fish sexual steroids, regulate the respiratory burst activity of acidophilic granulocytes obtained from the head-kidney (the bone marrow equivalent in fish) and the intracellular accumulation of IL-1beta by these cells. It is likely, therefore, that IL-1beta produced by testicular acidophilic granulocytes regulates important functions of the testis in fish.

Structure of the amphibian mesonephric tubule during ontogenesis in Rana ridibunda L. tadpoles: early ontogenetic stages, renal corpuscle formation, neck segment and peritoneal funnels.

The morphological changes produced in the mesonephric tubule during ontogenesis, not previously reported in amphibians, are described in Rana ridibunda tadpoles using light and electron microscopic methods. The rudimentary nephron units do not develop synchronously along the subperitoneal nephrogenic ridged cord. The first signs of morphogenesis are the presence of round euchromatinic nuclei and mitotic figures. The subsequent developmental stages are characterized by detachment of the rudimentary nephrons from the nephrogenic cord. Renal corpuscle formation is characterized by glomerular expansion, differentiation of large fenestrated capillaries and the presence of a discrete mesangium and a small capsular space. Interstitial capillaries next to the renal corpuscle rudiments appear to induce invagination and differentiation of the capsular epithelium. Developing podocytes were cuboidal undifferentiated epithelial cells with scarce primary processes and with an extensive part of the cell surface lying flat on the glomerular basement membrane. These features reflect low or no glomerular filtration during nephron development. The ciliated neck segment and peritoneal funnels show similar structural features. The latter were not physically connected with the nephrons, but opened into renal blood vessels. Involutive peritoneal funnels were observed.

Identification of mammosomatotropes, growth hormone cells and prolactin cells in the pituitary gland of the gilthead sea bream (Sparus aurata L., Teleostei) using light immunocytochemical methods: an ontogenetic study.

Growth hormone (GH) and prolactin (PRL) immunoreactivities in the adenohypophysis of Sparus aurata specimens from newly hatched until 48-months-old were detected using the peroxidase-antiperoxidase method. GH cells and PRL cells, and cells that were immunoreactive to both GH and PRL antisera, called mammosomatotropes (MS cells), were found. This is the first report on the identification of MS cells in fish, which were found in newly hatched and older larvae and juvenile specimens. GH and PRL cells appeared from two days after hatching. MS cells were first located in the central region of the adenohypophysis and afterwards in the rostral pars distalis. The GH cells were first identified in the dorsal and ventral areas of the middle-posterior part, and the PRL cells in the ventral region of the middle-anterior part. Later, during development, the sequence of appearance of the GH cells was proximal pars distalis, pars intermedia and rostral pars distalis, while for the PRL cells sequence was rostral pars distalis, proximal pars distalis and pars intermedia. This expansion pattern could be due to a GH- and PRL-cell migration although independent cell differentiation may occur in each region. The present results suggest that GH and PRL cells arise from MS cells at the outset of pituitary development, while MS cells proceed from PRL cells in old larvae and juveniles.

Ontogeny of immunoreactive somatolactin cells in the pituitary of gilthead sea bream (Sparus aurata L., Teleostei).

This is the first report on the identification of somatolactin (SL) cells during the early developmental stages of the teleost fish Sparus aurata. The SL cells were followed from newly hatched until 46 months. SL cells were immunocytochemically identified at light microscopical level with anti-cod SL in the developing pituitary using the peroxidase-antiperoxidase method. SL cells first appeared in newly hatched specimens, in which the pituitary gland lacked the neurohypophysis. They were scarce and located from the middle to the posterior region of the adenohypophysis. As the fish developed, the cells progressively increased in number and surrounded the developing neurohypophysis, which could be distinguised from 12-day-old larvae onwards in the caudal region of the gland. From 51 days onwards, SL cells were found in a discontinuous layer surrounding the neurohypophysis branches that entered the pars intermedia as clustered or isolated cells among non-SL-immunoreactive cells of the pars intermedia, and in the proximal pars distalis. The somatolactin-immunoreactive cells are periodic acid-Schiff-positive only in the adult stages. These data confirm, previous findings concerning the presence of two molecular forms of SL, glycosylated and nonglycosylated, in this species.

Early organization of the pituitary gland in Sparus aurata L. (Teleostei). An ultrastructural study.

The cell organization of the pituitary gland and the relationship between neurohypophysis and adenohypophysis in early developmental stages of the gilthead sea bream, Sparus aurata, were studied by electron microscopy. In newly hatched larvae, the pituitary gland was embedded in the ventral floor of the diencephalon and separated from the hypothalamus by a continuous basal lamina. Elongated mesenchymal cells next to the ventral surface were observed. At this stage, there was no neurohypophysis and the adenohypophysis consisted of undifferentiated endocrine cells with small scarce secretory granules and a few stellate cells, with no distinctive zonation. An incipient neurohypophysis was present in 1-day-old larvae. The first evagination of the neurohypophysis into the adenohypophysis were observed in 2-day-old larvae and developed progressively with age, being deeper in the caudal zone. Two regions in the adenohypophysis, one anterior--the presumptive pars distalis--and one posterior--the presumptive pars intermedia--were found in 2-day-old larvae. Three regions (rostral and proximal pars distalis and pars intermedia) were clearly distinguishable in 4-day-old larvae. The ultrastructural features of the pituitary endocrine cells varied during gland differentiation, with the secretory granules gradually increasing in number and size, accompanying organelle development. Nevertheless, even in the oldest larvae studied (65 days), undifferentiated cells similar to those in the earliest stages were observed. The first blood vessels appeared in the neurohypophysis around 16 days after hatching. During early development, the pituitary gland progressively emerged from the ventral floor of the brain. By 16 days, the principal pattern of the pituitary gland architecture appeared to be established.

Immunocytochemical and ultrastructural characterization of somatolactin cells from the gilthead sea bream (Sparus aurata L., Teleostei): an ontogenic study (from newly hatched to adults).

For the first time, somatolactin (SL) cells have been ultrastructurally identified and characterized during the ontogeny of gilthead sea bream, Sparus aurata, using specimens ranging in age from hatching to 15 months. The SL cells were identified by an immunogold method using anti-cod SL serum. The SL-immunoreactivity was mostly located on the secretory granules of the cells, although some vesicles of variable size and shape with a medium electron-dense content, and some irregular secretory granules and polymorphic or very irregular masses that can arise from the fusion of several secretory granules, also presented immunogold labeling. In adults, the SL cells were mainly found in the pars intermedia, where they were organized in discontinuous cell cords lying against the neurohypophysis or surrounding the neurohypophyseal branches. Some SL cells, however, appeared isolated or in small groups in the pars intermedia, in the proximal pars distalis and, rarely, in the rostral pars distalis. The SL cells were variable in shape, with processes directed towards the neurohypophysis or blood vessels, or intermingling among other adenohypophyseal cells. The secretory granules were mostly round, although some were oval, bilobate or pear-shaped, with a homogeneous, very electron-dense content and a narrow, dense or clear, halo. Different SL cell populations can be distinguished according to secretory granule size. Our findings indicate that SL is stored in the secretory granules and released by exocytosis. SL cells showing involutive features were only found in adults. SL cells can be ultrastructurally identified in one-day-old larvae although similar characteristics to those found in adults can be positively identified only after 4 days. Secretory granules increased in number, size and heterogeneity during development. Synaptic-like structures between axon terminals of the neurohypophysis and the SL cells were found in larvae from one-day-old onwards. In juveniles of 118 days of age, two different populations of secretory granules (immunogold-labeled and non-immunogold-labeled) can be found in the same or different SL cells, findings that suggest the existence of two different molecular forms of SL at this age. There was a clear increase in the complexity of the pituitary gland and in the heterogeneity of the SL cells during development, the latter observation probably reflecting different functional cell stages or production of SL molecules.

The adenohypophysis of Mediterranean yellowtail, Seriola dumerilii (Risso, 1810): an immunocytochemical study.

The adenohypophysis (ADH) of the Mediterranean yellowtail was studied using the peroxidase-antiperoxidase technique. Human corticotropin (ACTH) (1-24)-immunoreactive (ir) cells were found bordering the neurohypophysis (NH) and salmon prolactin (PRL)-ir cells were arranged in thick cords, both in the rostral pars distalis (RPD). Gonadotropin (GTH)-, thyrotropin (TSH)- and growth hormone (GH)-ir cells were observed in the proximal pars distalis (PPD). Anti-chum salmon GTH I and anti-chum salmon GTH II immunostained the same cells in the outermost part of the ADH at the level of the PPD and the PI. In addition to these cells, some cells grouped in the inner areas of the posterior PPD were revealed by catfish alpha, beta-GTH antiserum. Human beta-TSH-ir cells formed small groups and discontinuous strands in the PPD often in contact with the NH. Tilapia GH-ir cells formed cords mainly surrounding the NH in the central PPD, while cod somatolactin- and alpha MSH-ir cells mainly surrounded the NH branches in the PI.

Effects of radiation on rabbit thyroid gland ultrastructure.

The ultrastructure of irradiated rabbit thyroid gland (0 Gy, as control, 4 Gy, 8 Gy, 12 Gy, 16 Gy and 20 Gy X-ray) is studied. The thyroid gland of the control group shows ultrastructural features similar to those described in other mammals. Modifications in follicular ultrastructure and interfollicular connective tissue ultrastructure as well as the presence of small thyroid follicles, probably neoformed, are found to be dose-dependent. Follicular epithelium varies from simple cuboidal to simple squamous, the latter being characteristic of the highest X-ray dose. Follicular cells show numerous cytoplasmic light vesicles and a very extensive rough endoplasmic reticulum consisting of vacuolated cisternae. Sloughed cells are present at 8 Gy. A well-developed interstitial glandular tissue consisting of extensive extracellular matrix, abundant collagen fibres and dilated, irregularly outlined capillaries are also present.

Specific and non-overlapping functions of testosterone and 11-ketotestosterone in the regulation of professional phagocyte responses in the teleost fish gilthead seabream.

Sex hormones, both estrogens and androgens, have a strong impact on immunity in mammals. In fish, the role of androgens in immunity has received little attention and contradictory conclusions have been obtained. However, it is well known that sex steroids are involved in fish growth, osmoregulation and gonad remodelation. In this study, we examine the in vitro effects of testosterone and 11-ketotestosterone, the two main fish androgens, on the professional phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). Although both testosterone and 11-ketotestosterone failed to modulate the respiratory burst of seabream phagocytes, testosterone but not 11-ketotestosterone was able to increase the phagocytic ability of non-activated phagocytes. Curiously, 11-ketotestosterone was more powerful than testosterone at inducing the expression of its own receptor, namely androgen receptor b (ARb), in acidophilic granulocytes (AGs), but none of them affected the basal ARb expression levels in macrophages (MØ). Furthermore, although physiological concentrations of testosterone exerted a pro-inflammatory effect on both AGs and MØs, 11-ketotestosterone showed an anti-inflammatory effect in AGs and a strong pro-inflammatory effect in MØs. Interestingly, both androgens modulated the expression of toll-like receptors in these two immune cell types, suggesting that androgens might regulate the sensitivity of phagocytes to pathogens and damage signals. Testosterone and 11-ketotestosterone have a competitive effect, at least, on the modulation of the expression of some genes. Therefore, our results show for the first time a non-overlapping role for testosterone and 11-ketotestosterone in the regulation of professional phagocyte functions in fish.

In situ forming microparticle implants for delivery of sex steroids in fish: Modulation of the immune response of gilthead seabream by testosterone.

Current knowledge on the sensitivity of marine fish to androgenic environmental chemicals is limited, despite the growing interest in the effects of endocrine disrupting chemicals. To study in vivo the effects of testosterone (T) on the fish immune response, we used a microencapsulation implant technique, the in situ forming microparticle system, containing 1 mg T/kg body weight (T-ISM), in adult specimens of gilthead seabream (Sparus aurata L.), a species of great economic interest. We demonstrated that implants themselves (without T) have no significant effect on most of the parameters measured. In T-ISM implanted fish, T serum levels reached supraphysiological concentrations accompanied by a slight increase in 11-ketotestosterone and 17ß-estradiol levels 21 days post-implantation (dpi). Liver and head-kidney samples were processed 7 and 21 dpi to assess T-ISM effect on (i) the mRNA expression of genes involved in the metabolism of steroid hormones and in the immune response, and (ii) phagocyte activities. The expression profile of cytokines, chemokines and immune receptors was altered in T-ISM implanted animals that showed an early pro-inflammatory tendency, and then, a mixed pro-/anti-inflammatory activation during longer exposure. Furthermore, the enhancement of phagocytic activity and the production of reactive oxygen species by leukocytes 21 dpi in T-ISM implanted specimens suggest fine modulation of the innate immune response by T. Taken together, these data demonstrate for the first time the feasibility of using ISM implants in an aquatic species, and provide new data on the role played by T on the immune response in fish.

Natural and synthetic estrogens modulate the inflammatory response in the gilthead seabream (Sparus aurata L.) through the activation of endothelial cells.

Sex steroids are known to deeply alter processes other than fish reproduction, including fish growth, intermediary metabolism, osmoregulation and immunity. We have previously reported that 17ß-estradiol (E(2)), the main fish estrogen, promotes the mobilization of acidophilic granulocytes from the head kidney, the bone marrow equivalent in fish, to the gonad in the bony fish gilthead seabream (Sparus aurata L.). The aim of this study was to investigate the effects of E(2) and 17α-ethinylestradiol (EE(2)), an endocrine disruptor with strong estrogenic effects commonly found in the aquatic environment, on the ability of gilthead seabream endothelial cells (ECs) to promote leukocyte infiltration. E(2) and EE(2) were seen to affect ECs in different ways. Thus, E(2) was able to increase the production of nitric oxide (NO) and up-regulate the expression of the key activation markers, interleukin-1ß, CC chemokine ligand 4, interleukin-8, E-selectin and matrix metalloproteinase 9, when used alone or combined with bacterial DNA. In contrast, EE(2) failed to affect NO release and reduced the up-regulation of the above genes promoted by bacterial DNA. Moreover, we found that leukocyte adhesion to ECs was enhanced by E(2) treatment. Collectively, these results suggest that estrogens modulate fish leukocyte trafficking during an inflammatory process by activating ECs.